In recent years, the worldwide research focus on the development of anti-HIV drugs has tested the viability of receptor-based drug design and has helped to focus on de novo drug design. The proteinase of the Human Immunodeficiency Virus, HIV-1 protease (Blundell et al., 1990, TIBS 15 425-430; Huff, J., 1991, J. Med. Chem. 34 2305-2314; Darke, P. L. and Huff, J. R., 1994, Adv. Pharm. 25 399-454; Debouck, C., 1992, AIDS Res. & Hum. Retrovir 8 153-164), is a conspicuous example of a receptor for which drug design methodologies have been applied with some success. The rational design of clinically effective inhibitors for this enzyme has remained elusive.
The Human Immunodeficiency Virus (HIV) now infects over 15 million people worldwide, including some 20,000 individuals in Australia. Drugs aimed at treating HIV infection are being rationally developed to target key regulatory proteins that are essential for the replication of HIV. One of these proteins, HIV-1 protease (HIVPR), is an aspartic protease (James, M. N. G. & Sielecki, A. R., 1989, in Biological Macromolecules & Assemblies (Jurnak, F. A. & McPherson, A. M., eds), Wiley, N.Y., Vol. 3, p. 413; Fitzgerald, P. M. & Springer, J. P., 1991, Ann. Rev. Biophys. Chem. 20 299-320) that acts late in the viral replicative cycle by processing polypeptides (Pr160 and Pr50) transcribed from the gag and pol genes. The protease is essential for assembly and maturation of infectious virions but becomes inactivated by a single mutation (Asp25) in the active site, resulting in immature, non-infective virus particles (Kohl et al., supra; Ashorn et al., Proc. Natl. Acad. Sci. U.S.A. 87 7472-7476; Lambert et al., 1992, Antimicrob. Agents. Chemother. 36 982-988). Since inhibitor binding to HIVPR can also prevent infection of immune cells by HIV, the protease is a valid target for chemotherapeutic intervention (Kohl, N. E., 1988, Proc. Natl. Acad. Sci. USA 85 4686-4690; Ashorn et al., supra; Lambert et al., supra). Inhibitors of HIVPR can be expected to be possible treatments for HIV-infections. Indications are that resistance is more difficult to develop against HIVPR inhibitors (Roberts et al., 1990, Science 248 358-631; Craig et al., 1991, Antiviral Res. 16 295-305; Muirhead et al., 1993, 9th Intl. Cong. AIDS (Berlin), Abs PO-B30-2199; Muirhead et al., 1992, Br. J. Clin. Pharm. 34 170-171; Roberts et al., 1992, Biochem. Soc. Trans. 20 513-516) than reverse transcriptase inhibitors (Tomasselli et al., 1992, Chimicaoggi 6-27), but resistance is still proving to be a major problem.
HIVPR is a homo-dimer, consisting of two identically folded 99 amino acid subunits that form a hydrophobic active site cavity. The C.sub.2 symmetry of the enzyme is a unique feature among aspartic proteinases. HIVPR is also characterised by two conformationally flexible flaps (one per subunit) which are able to close around the substrate. The three dimensional crystal structures of both recombinant and synthetic HIVPR have been reported for the enzyme as well as enzyme-inhibitor complexes (Tozser et al., 1992, Biochemistry 31 4793-4800; Swain et al., 1990, Proc. Natl. Acad. Sci. USA 87 8805-8809). The major difference between these enzyme conformations is in the location of the flaps and some residues in the hinge region. The amino acids of HIVPR that line the substrate-binding groove, which is 24 .ANG. long by 6-8 .ANG. diameter, are symmetrically disposed around the catalytic residues located near the centre of the active site.
The first approaches to developing inhibitors of HIVPR involved a combination of analogue-based and mechanism-based drug design that focused on the amino acid sequence of substrates for HIVPR. These inhibitors were based on the observed preference for proteolysis of substrates with a scissile hydrophobic-hydrophobic or aromatic-proline peptide bond (Griffiths, J. T., 1992, Biochemistry 31 5193-5200) and were both potent and selective for HIVPR. Aside from optimising the fitting of amino acid side chains into the corresponding binding pockets (Wlodawer, A. & Erickson, J. W., 1993, Ann. Rev. Biochem. 62 543-585 and references therein) that line the substrate-binding groove of HIVPR, inhibitor design must also take into account hydrogen bonding and electrostatic interactions which occur along the binding groove.
Roberts et al., 1992, Biochem. Soc. Trans. 20 513-516; Pharmaprojects AN 017782 9202. PJB Publications Ltd., Richmond, Surrey, U.K.,; Paessens et al., 1993, 9th Intl. Cong. AIDS (Berlin) Abs PO-A25-0591 & PO-A25-0611; Pharmaprojects AN 019149 9212 & AN 020519 9310, PJB Publications Ltd., Richmond, Surrey, U.K.; 206th ACS (Chicago), 1993, MEDI 138; 32nd ICAAC (Anaheim) 1992, Abs 315 & 1501; Getman et al., 1993, J. Med. Chem. 36 288-291; Alteri et al., 1993, Antimicrob. Agents. Chemother. 37 2087-2092; Pharmaprojects AN 020519 9310, PJB Publications Ltd., Richmond, Surrey, Kim et al., 1993, 9th Intl. Cong. AIDS (Berlin) Abs PO-A25-0622; Vacca et al., 1994, Proc. Natl. Acad. Sci. USA 91 4096-4100; Pharmaprojects AN 020180 9307, PJB Publications Ltd., Richmond, Surrey, U.K.; Jadhav et al., 9th Int. Cong. AIDS (Berlin) Abs PO-A25-585; Vacca et al., 9th Int. Cong. AIDS (Berlin) Abs PO-B26-2023; Young et al., 1992, J. Med. Chem. 35 1702-1709; Cohen et al., 1990, J. Med. Chem. 33 883-894; Thiasrivongs et al., August 1994, Proc. 10th Int. Conf. AIDS (Yokohama) Abs 322A; Pharmaprojects AN 018824 9209, PJB Publications Ltd. Richmond, Surrey, U.K.; Kiso et al., 1993, 9th Intl. Cong. AIDS (Berlin) Abs PO-A25-567; 32nd ICAAC (Anheim), 1992, Abs 317-318; Kageyama et al., 1993, Antimicrob. Agents Chemother 37 810-817; Mimoto et al., 1992, Chem. Pharma. Bull 40 2251-2253; Scrip-world Pharmaceutical News, 1992, 1633 p 26; The Blue Sheet, 1993 35 p 8; Pharmaprojects AN 014606 9108, PJB Publications Ltd. Richmond, Surrey, U.K.; Danner et al., 1993, 9th Intl. Cong. AIDS (Berlin) Abs WS-B2606; Kempf et al., 1991, Antimicrob. Agents Chemother 35 2209-2214; Kort et al., 1993, Antimicrob. Agents Chemother 37 115-119.26; Kempf et al., 1992, J. Org. Chem. 57 5692-5700; Kempf et al., 1993, J. Med. Chem. 36 320-330; Erickson et al., 1990, Science 249 527-533; Pharmaprojects AN 018825 9209, PJB Publications Ltd. Richmond, Surrey, U.K.; 8th Intl. Conf. AIDS (Amsterdam), 1992, Abs ThA1507; Melnick et al., 1994, Proc. 207th ACS National Meeting (San Diego) Abs MEDI-20; Reich, S. H. 1994, Proc. of New Advances in Peptidomimetics and Small Molecule Design (Philadelphia); Sheety et al., August 1994, Proc. 10th Int. Conf. AIDS (Yokohama) Abs 321A; Lam et al., 1994, Science 263 380-384; Grzesiek et al., 1994, J. Am. Chem. Soc. 116 1581-2 and Otto et al., August 1994; Proc. 10th Int. Conf. AIDS (Yokohama) Abs 320A describe some of the more established potent inhibitors of HIVPR in vitro. Reference may be made to Smith et al., 1994, Biorganic & Medicinal Chemistry Letters 4 No. 18 2217-2222 which refers to HIV proteases comprising conformationally constrained peptide based hydroxyethylamines with 17 to 19 membered macrocyclic ring systems. These inhibitors all require the decahydroisoquinoline ring at the C terminus and the large size of the ring system does not permit these compounds to specifically mimic the substrate(s) of HIV protease.
All of the inhibitors in the abovementioned references are potent inhibitors of infection of cultured human cells in vitro, although their potency is usually 1-2 orders of magnitude lower in cells than against HIVPR in vitro. This problem is also discussed in Rich et al., 1990, J. Med. Chem. 33 1285; Toth et al., 1990, Int. J. Peptide Protein Res. 36 544-550; Brinkworth et al., 1991, Biochem. Biophys. Res. Comm. 176 241-246 and Majer et al., 1993, Arch. Biochem. Biophys. 304 1-8. Most of these compounds are being or have been investigated by pharmaceutical companies in vitro and in vivo and as prospective anti-viral drugs.
In contrast to reverse transcriptase inhibitors referred to in Tomasselli et al., supra, and Petteway et al., 1991, TiPS 12 28-34; Clercq, E. D., 1987, TiPS 8 339-345 and Field, H. & Goldthorpe, S. E., 1989, TiPS 10 333-337, protease inhibitors are able to block HIV infection in chronically as well as acutely infected cells which is crucial for clinical efficacy (Roberts et al., 1990, supra; Craig et al., 1991, supra; Muirhead et al., 1993, supra; Muirhead et al., 1992, supra and Roberts et al., 1992, supra, for example.
All of the substrate-based protease inhibitors described in the abovementioned references which are potent inhibitors of HIV infection of cultured human cells in vitro suffer as drugs from a combination of pharmacodynamic and pharmacokinetic problems. These problems are also discussed in Field, H. & Goldthorpe, S. E., 1989, TiPS 10 333-337; Kageyama et al., 1992, Antimicrob. Agents Chemother. 36 926-933; Sandstrom, E. & Obert, B. 1993, Drugs 45 637-653 and PALLAS pKalc and PrologP available from Compudrug Chemistry Ltd., Hungary. These problems include:
(i) short serum half lives (t.sub.1/2) and high susceptibility to hydrolysis by degradative enzymes present in the blood stream, gut and cells; PA1 (ii) poor absorption, low water-solubility and oral bioavailability; and PA1 (iii) rapid liver clearance and biliary excretion. PA1 (i) they are subject to proteolysis or hydrolysis by peptidases and therefore do not reach cells infected with HIV-1; PA1 (ii) they do not have a stable receptor-binding configuration insofar as they are capable of many different conformations; or PA1 (iii) they are not anti-viral in nature, i.e. they do not prevent viral replication. PA1 X=--(CH.sub.2).sub.n -- where n=3-6 but is preferably 3, 4 or 5, alkyl of 1-6 carbon atoms inclusive of linear and branched chains as well as cycloalkyl --CH(OH)--CH(OH)--CH.sub.2 --, --CH(CO.sub.2 H)--CH.sub.2 --CH.sub.2 --, or --CH.sub.2 CONHCHR-- where R=D- or L- amino acids and especially Lys, Arg, His, Tyr, Phe, Glu, Gln, Ile, Val or Asp, PA1 Y=side chains of Asn or Ile or Val or Glu; or alkyl of 1-6 carbon atoms inclusive of linear and branched chains as well as cycloalkyl ##STR3## wherein P=H, alkyl, aryl, Oalkyl, Nalkyl Q=H, alkyl, aryl, Oalkyl, Nalkyl
To improve their stability and bioavailability, various synthetic modifications need to be made but if these become too sophisticated, the economic feasibility of drug production can be compromised.
Structural modifications, involving polar ionisable groups that lead to increased gastrointestinal absorption and plasma concentrations of a renin inhibitor (Rosenberg et al., 1981, J. Med. Chem. 34 469-471), are also being made to improve the pharmacological profile of protease inhibitors. This approach is discussed in Flentge et al., 1984, Proc. 206th ACS National Meeting (San Diego), MEDI-35.
A major strategy to reduce pharmacological problems is to develop inhibitors which either structurally or functionally mimic bioactive peptides but have reduced or no peptide character. Few non-peptide inhibitors have been reported to date. Some are described in Debouck, C., 1992, supra; Brinkworth, R. I. & Fairlie, D. P. 1992, Biochem. Biophys. Res. Commun. 188 624-630; Lam et al., 1994, supra; Grzesiek et al., 1994, supra; Otto et al., supra; Tung et al., August 1994, Proc. 10th Int. Conf. AIDS (Yokohama) Abs 426A; DesJairlais et al., 1990, Proc. Nat. Acad. Sci USA 87 6644-664; Rutenber et al., 1993, J. Bio. Chem. 268 15343-15346 and Saito et al., 1984, J. Biol. Chem. 269 10691-10698. This approach is described in Vacca et al., 1994, supra; Pharmaprojects AN 020180 supra; Jadhav et al., 1993, supra; Vacca et al., 9th Int. Cong. AIDS (Berlin) Abs PO-B26-2023; Young et al., 1992, supra; Tucker et al., 1992, supra and Bone et al., supra which refers to decrease in inhibitor size, which might theoretically minimise biliary excretion while at the same time increasing water solubility. However, such an approach has so far met with limited success.
However, from the foregoing, it will be appreciated that prior art HIV-1 protease inhibitors which are mostly peptides or peptide derived in character have suffered from a number of disadvantages which include: